coli B7A with L-cysteine to label the DNA. The discovery was based on the inability of nuclease P1 to cleave the phosphorothioate bond. Sequence specific phosphorothioate d(G PSA) and d(G PSG) were first detected in E. In this modification, the non-bridging oxygen of the backbone phosphate group is replaced by sulfur. The chemical nature of this unusual DNA modification was eventually found to be a phosphorothioate modification of the DNA backbone by Wang et al. This feeding experiment set up a link between the unusual DNA modification and sulfur. 35S signals were detected in the DNA from three Dnd + strains, but not in Dnd - mutant ZX1 or Streptomyces coelicolor. Total genomic DNAs were prepared and analyzed on agarose gel followed by Southern blotting. lividans, Streptomyces avermitilis NRRL8165, and Pseudomonas fluorescens Pf0-1 were selected to propagate in media containing 35SO4 2. were prompted to conduct the 35S labeling experiment. Based on the information that two genes involved in this modification are related to sulfur transfer (section 2), Zhou et al. The chemical nature of this unusual DNA modification is an intriguing question. The modifying reagents most probably acted post-replicatively on unmodified double-stranded DNA substrates. Meanwhile, no Tris-mediated scission was detected in single-stranded plasmid replication intermediates, supporting the post-replicative mechanism. later revealed that the modification required a conserved consensus sequence, as well as flanking sequences with potential for secondary structure(s) (section 3). Based on these observations, it was proposed that the DNA degradation was the consequence of a site-specific modification, which suffered cleavage by oxidative Tris resulting in degradation during electrophoresis. Alternatively, non-degradative electrophoresis of the DNA could also be achieved in a different buffer such as Hepes. Thiourea can react with the Tris derivative and thus inhibits the DNA scission. lividans is intact, and the degradation only occurs during electrophoresis in the presence of oxidative Tris. then verified that the Dnd phenotype depends on the cleavage activity of an oxidative Tris derivative generated in the electrophoretic buffer adjacent to the anode. lividans underwent site-specific cleavage during electrophoresis, giving particular fragment profiles. For instance, both plasmid pIJ101 and pIJ303 from Dnd + S. The modification sites are not randomly distributed in DNA. demonstrated that such a Dnd phenotype was not due to nuclease contamination or improper in vitro genetic manipulation, but instead, an unusual DNA modification. The study of the physiological DNA phosphorothioation originated from an observation that an unusual DNA modification in Streptomyces lividans renders DNA susceptible to in vitro Tris-dependent double strand cleavage, resulting in a DNA degradation (Dnd) phenotype during conventional and pulsed-field gel electrophoresis. Discovery of phosphorothioation as an unusual post-replicative modification on the DNA backbone Here we summarize the discovery of this first reported physiological modification on the DNA backbone, and provide insights and perspectives into the biological functions of the phosphorothioate modification in prokaryotic physiology.ΔΆ. Interestingly, the gene sco4631, which code for ScoA3McrA, is unable to coexist with the dnd gene cluster in the same host, causing immediate cell death. Another type IV endonuclease, ScoA3McrA, was found to be capable of specifically recognizing as well as cleaving phosphorothioate modified DNA. Recently, a counterpart phosphorothioate-dependent restriction system capable of protection against the invasion of unmodified foreign DNA was discovered to maintain the genetic stability of the phosphorothioate modified host. The physiological phosphorothioate modification is widespread in bacteria and occurs in diverse sequence contexts and frequencies in different bacterial genomes, implying a significant impact on bacteria. Unlike any other DNA or RNA modification system, DNA phosphorothioation is the first-described physiological modification of the DNA sugar-phosphate backbone. The five elements of nitrogen, phosphorus, carbon, hydrogen, and oxygen had been regarded as the canonical composition of DNA until the discovery of phosphorothioation, with a sixth element, sulfur, identified as an additional naturally occurring constituent on the DNA backbone, as a sequence-selective, stereospecific post-replicative modification governed by the dnd gene cluster. They appear to fulfill all requirements necessary to maintain the genetic function of DNA. DNA molecules are polymers composed of basic repeating subunits of deoxyribonucleotides, which consist of the deoxyribose sugar, phosphate groups, and a nitrogenous base.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |